All of the diverse cells in a human body contain the same “library” of genetic information stored in the form of DNA molecules. Over the past 40 years, scientists have made considerable progress in understanding how the cell reads DNA to decode the information carried in our chromosomes. This process of gene expression is exquisitely regulated and drives the formation of all the differentiated specialized cell types within organs such as brain, breast and skeletal muscles. The experiments described in this proposal are directed at understanding the regulation of gene expression in both self-renewing and differentiating stem cells. These experiments represent a critical first step in the quest to propagate human embryonic stem cells and to derive differentiated cells from them for the purposes of disease therapy. In preliminary studies, our laboratory has identified a novel protein complex, SCC, required for activating genes needed to maintain stem cell self-renewal – an essential defining property of stem cells. Here we propose to characterize the molecular components of this complex as an important step in our efforts to reliably grow human stem cells in a reproducible manner. At the same time we hope to determine whether these same key proteins are also active in cancer cells which may complicate the use of stem cells in human therapeutic applications. Thus, regulatory factors such as SCC are potential targets for drugs aimed at increasing or decreasing the ability of stem cells to divide. A second major focus of the proposal is to understand the network of gene activity needed for the differentiation of dopamine-producing neurons, the brain cells that are lost in Parkinson’s disease. Our collaborators have devised a method to grow dopamine neurons from human embryonic stem cells in culture. Here we propose biochemical and molecular biological studies to identify the genes active in these dopamine neurons, with the goal of improving the efficiency of dopamine neuron culture and subsequently, the development of new therapies for Parkinson’s disease. Finally, we will investigate the regulatory proteins required for gene activity in muscle cells. We will use biochemical assays to understand the different activities of a key regulatory protein, TAF3, in immature and differentiated muscle cells, and will use cell culture strategies similar to those employed for dopamine neurons to differentiate muscle cells from human embryonic stem cells in culture. Many of the muscular dystrophies are the consequence of mutations in genes expressed specifically in muscle cells. The proposed studies could lead to strategies for drug design or treatment for these conditions.

Statement of Benefit to California:

The ultimate goal of these studies is the development of new therapies for diseases that are fundamentally the result of inappropriate levels of cell division and differentiation. The proposed experiments will determine how gene activity is controlled, either to maintain a renewing population of stem cells, or to direct differentiation of specific mature cell types implicated in human disease. For example, Parkinson’s disease and muscular dystrophies arise because the body is unable to replace damaged cells, dopamine neurons and muscle cells respectively. Conversely, breast cancer, another disease targeted by the proposed experiments, is the result of misregulated cell division and the failure of the newly produced cells to assume an appropriate location or function. Because all biological activities; cell division, differentiation and function, are the result of differential gene expression, an understanding of the gene regulatory networks controlling these processes will be crucial for drug development and testing. Thus, this proposal will not only advance the science of stem cells but also provide the technological platform for establishing new bio-pharma enterprises based on the development of disease intervention using original cell based assays for drug discovery . The proposed experiments will benefit the people of the State of California both directly and indirectly. In the short term, the research will support the training of four post-doctoral scholars and one graduate student, three lifelong California residents and two new residents who have moved here specifically to participate in this project. CIRM funding will also enable us to expand our collaboration with a European laboratory, bringing new stem cell technologies and expertise to the State. In the long term, the proposed work will likely reveal gene activities that are essential for the establishment, survival and maintenance of stem cells as well as differentiated cell populations. These studies will likely reveal previously unknown potential drug targets that will allow the screening of novel classes of drugs. For example, one possible target is the stem cell coactivator complex SCC. By increasing SCC activity, it may be possible to enhance the expansion of stem cell populations. Conversely, if SCC activity is necessary for the perpetuation of breast cancer stem cells, this would be an attractive target for drugs aimed at decreasing cancer cell potency. This project will also result in the refinement of the methods for engineering dopamine neurons and muscle cells. Such cells could be used directly in stem cell therapy. The possibility of culturing these cells in significant quantities under defined conditions in vitro, in combination with a detailed understanding of their gene regulatory networks, will also open up new ways to screen for drugs useful in the treatment of Parkinson’s disease or muscular dystrophy.